Instrumentation and Chemistry
Instrumentation and Analysis Software
We use an Applied Biosystems 3730s Genetic Analyser fitted with a 50 cm array and is capable of processing 48 samples per run. Our instrument can generate read lengths in excess of 1,000 QV20+ bases with an enhanced basecaller. The proprietary Kb basecaller used during data analysis, incorporates advanced algorithms that produce accurate sequence basecalls. The file output (.AB1) is Windows format. The sequence data is supplied to you as an electropherogram (trace file) together with a standard text file of the basecalls. Standard Chromatogram format (.scf) and Phred format (.phd.1) are both available on request.
PeakTrace, an optional enhanced basecaller (uses a more sophisticated algorithm to the Kb basecaller) is also available which may generate up to an additional two hundred QV20+ basecalls dependent on the quality of the input template DNA. This enhancement provides far greater resolution throughout the sequence read.
We strongly recommend that you perform a full manual edit of all your sequence data using the trace files as a standard research practise.
The cycle sequencing chemistry currently used by Micromon is the Applied Biosystems PRISM BigDye Terminator Mix (versions 3.1 and 1.1) incorporating the key enzyme AmpliTaq DNA polymerase - FS. Version 3.1 is the mix recommended for most sequencing applications and we can supply the terminator mix directly to you, significantly discounted from the Applied Biosystems list price.
While our sequence runs generate in excess of 1,000 QV20+ basecalls, using the Kb basecaller, around 900 bases are routinely called with quality values exceeding QV=20 (error probability of 1.0% or less), the majority exceeding QV=50 (error probability of 0.001% or less) using the BigDye v3.1 chemistry.
The quality of the basecalls (resolution) is significantly dependent on the purity of the starting DNA template. A reduction in final resolution and signal quality can be caused by the presence of RNA, salt, protein and other contaminating chemicals. These contaminants can be eliminated by effective template purification prior to cycle sequencing and a thorough post-reaction clean up. These are both key factors in the generation of long, highly resolved reads.
The Applied Biosystems Chemistry Guide is a comprehensive guide to Sanger sequencing and includes an comprehensive section on troubleshooting with actual examples. The Qiagen guide is still an excellent basic resource, although quite dated now. It is important to note that the amounts of BigDye Terminator mix recommended in the AB guide are not practical for the current AB instruments and the recommended amounts can be diluted by at least, one in eight.